Treatment and prevention of abnormal cellular proliferation

ABSTRACT

This invention provides a method for inhibiting or preventing the abnormal growth of cells, including transformed cells, by administering an effective amount of O-acylated heparin derivative. Abnormal growth of cells refers to cell growth independent of normal regulatory mechanism (e.g. loss of contact inhibition). This includes the abnormal growth of: (1) tumor cells (tumors); (2) benign and malignant cells of other proliferative disease in which aberrant cellular proliferation occurs; (3) aberrant smooth muscle cell proliferation, such as might occur following treatment for coronary atherosclerosis such as angioplasty or the insertion of a stent into an occluded vessel.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Ser. No. 60/553,800 filed Mar. 16, 2004, thecontent of which is herein incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made in part with U.S. Government support under grantnumber 5 RO1 HL39150-15 awarded by the National Institutes of Health.The U.S. Government has certain rights in this application.

FIELD OF INVENTION

The present invention is directed to a method of treating or preventingabnormal cellular proliferation.

BACKGROUND OF THE INVENTION

Atherosclerosis, a common form of arteriosclerosis, results from thedeposition of fatty substances, primarily cholesterol, and subsequentfibrosis in the inner layer (intima) of an artery, resulting in plaquedeposition on the inner surface of the arterial wall and degenerativechanges within it. The ubiquitous arterial fatty plaque is the earliestlesion of atherosclerosis and is a grossly flat, lipid-rich atheromaconsisting of macrophages (white blood cells) and smooth muscle fibers.The fibrous plaque of the various forms of advanced atherosclerosis hasincreased intimal smooth muscle cells surrounded by a connective tissuematrix and variable amounts of intracellular and extracellular lipid. Atthe luminal surface of the artery, a dense fibrous cap of smooth muscleor connective tissue usually covers this plaque or lesion. Beneath thefibrous cap, the lesions are highly cellular consisting of macrophages,other leukocytes and smooth muscle cells. As the lesions increase insize, they reduce the diameter of the arteries and impede bloodcirculation resulting in coronary heart disease, myocardial infarction(MI) and other serious complications.

Many therapies have been considered for the treatment ofatherosclerosis, including surgery and medical treatment. One potentialtherapy is percutaneous transluminal angioplasty (balloon angioplasty).More than 400,000 such procedures are performed each year in the UnitedStates. In balloon angioplasty, a catheter equipped with an inflatableballoon is threaded intravascularly to the site of the atheroscleroticnarrowing of the vessel. Inflation of the balloon compresses the plaqueenlarging the vessel.

While such angioplasty has gained wider acceptance, it suffers from twomajor problems, i.e., abrupt closure and restenosis. Abrupt closurerefers to the acute occlusion of a vessel immediately after or withinthe initial hours following a dilation procedure. Abrupt closure occursin approximately one in twenty cases and frequently results inmyocardial infarction and death if blood flow is not restored in atimely manner.

As many as 50% of the patients who are treated by balloon angioplastyrequire a repeat procedure within six months to correct a re-narrowingof the artery. Restenosis refers to such re-narrowing of an artery afteran initially successful angioplasty. Restenosis of the blood vessel isthought to be due to injury to the endothelial cells of the blood vesselduring angioplasty, or during inflation of the balloon catheter. Duringhealing of the blood vessel after surgery, smooth muscle cellsproliferate faster than endothelial cells resulting in a narrowing ofthe lumen of the blood vessel and starting the atherosclerotic processanew. In recent years, smooth muscle cell proliferation has beenrecognized as a major clinical problem limiting the long-term efficacyof coronary angioplasty.

In an effort to prevent restenosis of the treated blood vessel, thesearch for agents that can reduce or prevent excessive proliferation ofsmooth muscle cells have been the object of much research. (Theoccurrence and effects of smooth muscle cell proliferation after thesetypes of surgery have been reviewed, for example, in Ip, et al., (June1990) J. Am. College of Cardiology 15:1667-1687, and Faxon, et al.(1987) Am. J. of Cardiology 60: 5B-9B.). Such compounds have foundlittle if any practical success. There therefore exists a need toidentify and successfully administer compounds that inhibit smoothmuscle cell proliferation.

An alternative to angioplasty is the placement of endovascular stents inthe occluded blood vessel. Placement of a stent at such a site, shouldmechanically block abrupt closure and delay restenosis (Harrison'sPrinciples of Internal Medicine, 14^(th) Edition, 1998). Of the variousprocedures used to overcome restenosis, stents have proven to be themost effective. Stents are metal scaffolds that are positioned in thediseased vessel segment to create a normal vessel lumen. Placement ofthe stent in the affected arterial segment prevents recoil andsubsequent closing of the artery. By maintaining a larger lumen thanthat created using balloon angioplasty alone, stents reduce restenosisby as much as 30%. Despite their success, stents have not eliminatedrestenosis entirely. (Suryapranata et al. 1998. Randomized comparison ofcoronary stenting with balloon angioplasty in selected patients withacute myocardial infarction. Circulation 97:2502-2502).

Unfortunately, the use of such stents are limited by direct (subacutethrombosis) or indirect (bleeding, peripheral vascular complications)complications. After stent implantation the patients are threatened withstent thrombosis until the struts of the stent are covered byendothelium. Thus, an aggressive therapy using anticoagulation and/orantiplatelet agents is necessary during this period of time. While thesetherapies are able to decrease the rate of stent thrombosis, they arethe main source of indirect complications.

In addition to coronary artery occlusion, narrowing of the arteries canoccur in other vessels. Examples include the aortoiliac, infrainguinal,distal profunda femoris, distal popliteal, tibial, subclavian andmesenteric arteries. The prevalence of peripheral artery atherosclerosisdisease (PAD) depends on the particular anatomic site affected as wellas the criteria used for diagnosis of the occlusion. Rates of PAD appearto vary with age, with an increasing incidence of PAD in olderindividuals. Data from the National Hospital Discharge Survey estimatethat every year, 55,000 men and 44,000 women had a first-listeddiagnosis of chronic PAD and 60,000 men and 50,000 women had afirst-listed diagnosis of acute PAD. Ninety-one percent of the acute PADcases involved the lower extremity. The prevalence of comorbid coronaryartery disease (CAD) in patients with PAD can exceed 50%. In addition,there is an increased prevalence of cerebrovascular disease amongpatients with PAD.

PAD can be treated using percutaneous transluminal balloon angioplasty(PTA). The use of stents in conjunction with PTA decreases the incidenceof restenosis. However, the post-operative results obtained with medicaldevices such as stents do not match the results obtained using standardoperative revascularization procedures, i.e., those using a venous orprosthetic bypass material. (Principles of Surgery, Schwartz et al.eds., Chapter 20, Arterial Disease, 7th Edition, McGraw-Hill HealthProfessions Division, New York 1999).

Preferably, PAD is treated using bypass procedures where the blockedsection of the artery is bypassed using a graft. (Principles of Surgery,Schwartz et al. eds., Chapter 20, Arterial Disease, 7th Edition,McGraw-Hill Health Professions Division, New York 1999). The graft canconsist of an autologous venous segment such as the saphenous vein or asynthetic graft such as one made of polyester, polytetrafluoroethylene(PTFE), or expanded polytetrafluoroethylene (ePTFE). Restenosis andthrombosis, however, remain significant problems even with the use ofbypass grafts. For example, the patency of infrainguinal bypassprocedures at 3 years using an ePTFE bypass graft is 54% for afemoral-popliteal bypass and only 12% for a femoral-tibial bypass.

Consequently, there is a significant need to improve the performance ofboth stents and synthetic bypass grafts in order to further reduce themorbidity and mortality of CAD and PAD.

With stents, the approach has been to coat the stents with variousanti-thrombotic or anti-restenotic agents in order to reduce thrombosisand restenosis. For example, impregnating stents with radioactivematerial appears to inhibit restenosis by inhibiting migration andproliferation of myofibroblasts. (U.S. Pat. Nos. 5,059,166, 5,199,939and 5,302,168). Irradiation of the treated vessel can pose safetyproblems for the physician and the patient. In addition, irradiationdoes not permit uniform treatment of the affected vessel.

Numerous attempts to develop stents with a local drug-distributionfunction have been made, most of which are variances of the so calledstent graft, a metal stent covered with polymer envelope, containing amedicament. It would be of benefit to coat a stent with a compoundcapable of diminishing or eliminating restenosis.

Unlike the unwanted smooth muscle cell proliferation seen in restenosis,cellular proliferation is a normal ongoing process in all livingorganisms and is one that involves numerous factors and signals that aredelicately balanced to maintain regular cellular cycles.

When normal cellular proliferation is disturbed or somehow disrupted,the results can be inconsequential or they can be the manifestation ofan array of biological disorders. Disruption of proliferation could bedue to a myriad of factors such as the absence or overabundance ofvarious signaling chemicals or presence of altered environments. Somedisorders characterized by abnormal cellular proliferation includecancer, abnormal development of embryos, improper formation of thecorpus luteum, difficulty in wound healing as well as malfunctioning ofinflammatory and immune responses.

Cancer is characterized by abnormal cellular proliferation. Cancer cellsexhibit a number of properties that make them dangerous to the host,often including an ability to invade other tissues and to inducecapillary ingrowth, which assures that the proliferating cancer cellshave an adequate supply of blood. One of the defining features of cancercells is that they respond abnormally to control mechanisms thatregulate the division of normal cells and continue to divide in arelatively uncontrolled fashion until they kill the host.

It is clear that aberrant cellular proliferation plays a major role inthe formation and progression of a cancer. If this abnormal orundesirable proliferative activity could be repressed, inhibited, oreliminated, then the tumor, although present, would not grow. In thedisease state, prevention of abnormal or undesirable cellularproliferation could slow or abate the progression of cancer.Additionally, compounds that could induce apoptosis of abnormallyproliferating cells would be especially beneficial for complete removalor elimination of malignant cells, helping to reduce relapses. Therapiesdirected at control of the cellular proliferative processes could leadto the abrogation or mitigation of such malignancies.

Pulmonary hypertension is caused largely by an increase in pulmonaryvascular resistance and is classified clinically as either primary orsecondary. Secondary pulmonary hypertension, the more common form, isgenerally a result of (1) chronic obstructive or interstitial lungdisease; (2) recurrent pulmonary emboli; (3) liver disease; or (4)antecedent heart disease. Primary pulmonary hypertension is diagnosedonly after all known causes of increased pulmonary pressure areexcluded.

At the moment there is no successful cure for pulmonary hypertension.Administration of vasodilatating drugs has not proved to be useful inpatients suffering from pulmonary hypertension. The prognosis is poor,with a median survival time of about 3 years.

Pulmonary fibrosis can occur in response to known stresses such asasbestos or silica but most is idiopathic. There is a spectrum ofidiopathic fibrosis but most kinds are fatal in 3-5 years. At present,there is no effective therapy for most cases.

What is needed therefore is a composition and method which can inhibitabnormal or undesirable cellular proliferation, especially the growth ofsmooth muscle cells after angioplasty, stent placement, pulmonaryhypertension, pulmonary fibrosis or the proliferation of malignantcells. The composition should be able to overcome the activity ofendogenous growth factors in premetastatic tumors and inhibit smoothmuscle cell proliferation during restenosis. Finally, the compositionand method for inhibiting cellular proliferation should preferably benon-toxic and produce few side effects.

Heparin is a glycosaminoglycan that was first described by McLean in1916 and has been used clinically as an anticoagulant for more than 50years [McLean, Circulation 19, 75-78 (1959)]. Members of theglycosaminoglycan family include hyaluronan, heparan sulfate, dermatansulfate, and chondroitin sulfate. Beyond its well-recognizedanticoagulant activity, heparin has other activities. The antimetastaticactivity of heparin has been known for some time (see, for example,Drago, J. R. et al., Anticancer Res., 4(3), 171-2, 1984).

Unfortunately, native and currently described modified heparins areextremely anticoagulant. Their anticoagulant properties are such thatdoses effective in the treatment of malignancies and anti-proliferativedisorders are not attainable. It has therefore been suggested thataltering the chemical structure of heparin might decrease theanticoagulant properties of heparin while maintaining its otherimportant biological activities, such as its antimetastatic activity(Barzu et al., J. Med. Chem, 1993, 36, pg. 3546-3555).

Low molecular weight heparins have shown promise in reducinganticoagulation while maintaining their antimetastatic activity. Forexample, when compared to unmodified heparin, 2-O-desulfated and3-O-desulfated heparins had reduced anticoagulant activities, butpreserved their angiostatic, anti-tumor and anti-metastatic properties(Masayuki et al., U.S. Pat. No. 5,795,875 (1997); Lapierre et al.,Glycobiology 6, 355-366 (1996)]. Nevertheless, the use of currentlyavailable heparins and heparin derivitives for the treatment of abnormalcellular proliferative disorders is not practical due to their markedanticoagulant and antithrombotic activities.

O-acylated heparins have been described. These molecules have very lowanticoagulative effects in vitro, yet retain activity against HIV-1 and2 induced cytopathicity (Barzu et al., J. Med. Chem, 1993, 36, pg.3546-3555).

A chemically modified heparin that can be used to treat and/or preventabnormal cellular proliferative disorders is needed. Such a compoundshould have minimal anticoagulant properties while maintainingantiproliferative properties. The anticoagulative properties of thecompound must not limit its use in the clinical setting.

SUMMARY

This invention provides a method for inhibiting or preventing theabnormal growth of cells, including transformed cells, by administeringan effective amount of O-acylated heparin derivative. Abnormal growth ofcells refers to cell growth independent of normal regulatory mechanism(e.g. loss of contact inhibition). This includes the abnormal growth of:(1) tumor cells (tumors); (2) benign and malignant cells of otherproliferative disease in which aberrant cellular proliferation occurs;(3) aberrant smooth muscle cell proliferation, such as might occurfollowing treatment for coronary atherosclerosis such as angioplasty orthe insertion of a stent into an occluded vessel. The O-acylated heparinderivative is preferably an O-hexanoylated heparin derivative or anO-butanoylated heparin derivative. In a preferred embodiment, theo-acylated heparin is weakly anticoagulant as compared to non-chemicallymodified heparins.

One embodiment of the present invention provides a method for inhibitingor preventing tumor growth by administering an effective amount of ano-acylated heparin, to a subject, e.g. a mammal (and more particularly ahuman) in need of such treatment. In particular, this invention providesa method for inhibiting the growth of malignant cells by theadministration of an effective amount of an O-acylated heparin. In apreferred embodiment, the methods are directed toward the treatment orprevention of lung and colon cancer. Preferably, the O-acylated heparinderivative is an O-hexanoylated or an O-butanoylated heparin derivative

In another embodiment, the invention provides a method of preventingabnormal smooth muscle cell proliferation. The method comprisespresenting an O-acylated heparin near or into a site of abnormal smoothmuscle cell proliferation. In a preferred embodiment, the methods aredirected toward the prevention of smooth muscle cell proliferation asoccurs in restenosis. The methods are used to prevent restenosis thatoccurs following angioplasty or vascular stent placement. Alternatively,the methods of the current invention are used to prevent restenosisfollowing coronary artery stent placement, peripheral artery stentplacement, or cerebral artery stent placement.

In yet another embodiment, the invention provides a medical devicecoated with the heparin composition, e.g., a stent for implantation in ablood vessel. The stent of the invention comprises a coating containingan O-acylated heparin and preferably, an O-hexanoylated orO-butanoylated heparin derivative. In one embodiment, the stent iscoated with an O-acylated heparin and one or more compounds selectedfrom the group consisting of a polymer, fiber polymer, polyurethane,silicone rubber elastomer, drug, hydrogel, or other acceptable compoundor carrier known to those of skill in the art. Other medical devicessuch as catheters may also be coated with the O-acylated heparin.

Finally, the invention provides a method of treating pulmonaryhypertension and pulmonary fibrosis. The methods of the presentinvention provide treating a subject with a therapeutic amount of ano-acylated heparin, preferably a O-hexanoylated or O-butanoylatedheparin derivative. In one embodiment, the invention provides for thetreatment of primary pulmonary hypertension. In another embodiment, theinvention provides for the treatment of secondary pulmonaryhypertension. In a final embodiment, the invention provides for thetreatment of pulmonary fibrosis.

DESCRIPTION OF FIGURES

FIG. 1 shows the preparation of O-acylated heparin derivatives.

FIG. 2 shows the results of ¹H NMR spectroscopy, where approximately 10mg of each sample was exchanged by lyophilization three times from 0.5ml portions of 99.9% 2H₂O before being redissolved in ²H₂O for NMRanalysis. Chemical shifts are reported relative to TMS at 0.00 ppm. Thedegree of substitution (O-acylation) was determined from the ratio ofthe integrated area of the peaks assigned to the aliphatic methylprotons of the hexanoyl group (0.753 ppm) to the anomeric proton ofIdoA2S (5.092 ppm).

FIG. 3 shows the structures of O-hexanoylated heparin and O-butanoylatedheparin.

FIG. 4 shows that hexanoylated heparin (HHP) significantly inhibitedpulmonary artery smooth muscle cell proliferation in vivo.

FIG. 5 shows that hexanoylated heparin (HHP) significantly inhibited thedevelopment of pulmonary hypertension induced by hypoxia in the piglung.

FIG. 6 shows a comparison of tumor growth in SCID mice treated withvarious doses of native heparin (UHP) and butanoylated heparin (BHP).Butanoylated heparin significantly inhibited A549 (non-small cell lungcarcinoma) cell tumor growth in a dose dependent manner.

FIG. 7 shows a comparison of tumor weights from SCID mice treated withnative heparin and butanoylated heparin. Butanoylated heparin (BHP)significantly decreased A549 (non-small cell lung carcinoma) cell tumorweight in a dose dependent manner.

FIG. 8 shows percent inhibition of tumor growth in SCID mice treatedwith native heparin and butanoylated heparin. Butanoylated heparin (BHP)significantly inhibited A549 (non-small cell lung carcinoma) cell tumorgrowth in a dose dependent manner.

FIG. 9 shows tumor growth in SCID mice treated with native heparin andbutanoylated heparin. Butanoylated heparin (BHP) significantly inhibitedDMS79 (small cell lung carcinoma) cell tumor growth in a dose dependentmanner.

FIG. 10 shows tumor weight from SCID mice treated with native heparinand butanoylated heparin. Butanoylated heparin (BHP) significantlydecreased DMS79 (small cell lung carcinoma) cell tumor weight in a dosedependent manner.

FIG. 11 shows tumor growth in SCID mice treated with native heparin andbutanoylated heparin. Butanoylated heparin (BHP) significantly inhibitedHCT116 (colon cancer) cell tumor growth in a dose dependent manner.

FIG. 12 shows tumor growth in SCID mice treated with native heparin andbutanoylated heparin. Butanoylated heparin (BHP) significantly inhibitedHCT116 (colon cancer) cell tumor growth in a dose dependent manner.

FIG. 13 shows coagulation time for various heparin compounds. Nativeheparin (UPJ HP) increased coagulation time in a dose dependent manner.Butanoylated heparin had no significant effect on coagulation time.

FIG. 14 shows histology of A549 cell tumor tissue grown in SCID mice.Hemorrhage was detected in mice treated with 100 mg/kg native,unfractionated heparin only.

FIG. 15 shows histology of heart from SCID mice bearing A549 cell tumor.No significant pathological change was observed in any group.

FIG. 16 shows histology of kidney from SCID mice bearing A549 celltumor. No significant pathological change was observed in any group.

FIG. 17 shows histology of liver from SCID mice bearing A549 cell tumor.No significant pathological change was observed in any group.

FIG. 18 shows histology of lung from SCID mice bearing A549 cell tumor.No significant pathological change was observed in any group.

FIG. 19 shows apoptosis index in different treatment groups. Heparinsignificantly induces apoptosis in tumor grown in SCID mice.

FIG. 20 shows expression of p27/KIP1 gene product in the A549 cell tumortissue grown in SCID mice treated with heparins. BHP significantlyinhibited p27/KIP1 in a dose dependent manner.

FIG. 21 shows expression of Rb gene product in the A549 cell tumortissue grown in SCID mice treated with heparins. BHP decreased Rb geneexpression in a dose dependent manner although to a lesser effect thanon p27/KIP1.

FIG. 22 shows expression of E2F1 protein in A549 cell tumor tissue grownin SCID mice treated with heparins. BHP significantly inhibited E2F1protein expression in a dose dependent manner.

FIGS. 23A and 23B show diagrams of a stent. FIG. 23A, a stent; FIG. 23B,an end view of the stent of FIG. 23A

FIG. 24 shows a diagram of a stent having a second coating formed on theouter surface.

DETAILED DESCRIPTION

The present invention is directed generally to compositions and theiruse in the therapy and prevention of abnormal cellular proliferativedisorders, such as cancer, (i.e. lung and colon cancer), restenosis(following angioplasy, vascular stent placement, coronary artery stentplacement, periphaeral artery stent placement, or cerebral artery stentplacement), pulmonary hypertension (primary or secondary), and pulmonaryfibrosis. The administration of therapeutic levels of the O-acylatedheparin derivatives result in a decrease, cessation, or prevention ofthe abnormal cellular proliferation.

O-Acylated Heparin

As described further below, compositions useful in the present inventioninclude, but are not restricted to, O-acylated heparins, particularlyO-hexanoylated heparin derivatives and O-butanoylated heparinderivatives.

O-acylated heparins are prepared using any of a variety of well knownsynthetic and/or recombinant techniques, an example of which is furtherdescribed below. Furthermore, O-acylated heparins, useful in the presentinvention, have been described in Barzu et al., J. Med. Chem, 1993, 36,3546-3555 and U.S. Pat. No. 4,990,502 (Lormeau et al.). The structure ofthe O-acylated heparin derivatives used in the present invention areshown in FIG. 3. Preferably, the major disaccharide units (m) vary fromabout 4 to about 14. Most preferably the major disaccharide units (m)vary from about 7 to about 9. In the O-hexanoylated derivative,R═CH₃(CH₂)⁴⁻ in the O-butanoylated derivative, R═CH₃(CH₂)²⁻.

Low-molecular weight heparins (LMWHs) are fragments of conventionalheparin. LMWHs were developed to provide more selective inhibition ofenzyme function and reduce adverse effects. Heparin fragmentationproduces products which maintain activity against factor X_(a) andrelease antithrombotic factors, but have significantly less activityagainst factor II_(a). As a result, treatment with LMWHs providesantithrombotic effects with less anticoagulant effect, lessening therisk of hemorrhage. However, in the generic sense, LMWHs have not provenbeneficial in the treatment of cancer due to their high anticoagulantactivity.

Administration of Compounds

The heparins of the present invention can be administered via anymedically acceptable means which is suitable for the compound to beadministered, including oral, rectal, topical, parenteral (includinginhaled, subcutaneous, intramuscular and intravenous) administration, orby coated stent, coated graft, or coated catheter.

Effective doses for heparin-like substances are well known to those ofskill in the art. Generally, for heparin-like substances, an effectivedose is that which maintains the anti-X_(a) level between 0.5 and 1.0units/ml. This range has been shown to optimize antithrombotic activitywhile avoiding adverse effects.

The total daily dose may be given as a single dose, multiple doses,e.g., two to six times per day, or by intravenous infusion for aselected duration. Dosages above or below the range cited above arewithin the scope of the present invention and may be administered to theindividual patient if desired and necessary. If discrete multiple dosesare indicated, treatment might typically be 4-6,000 units of a compoundgiven 4 times per day or if given continuously, as is more often thecase, then a loading dose of 80 units/kg followed by 18 units/kg/hr(Rascke R A, Reilly B M, Guidry J R, et al. The weigh based heparindosing nomogram compared with a “standard care” nomogram: A randomizedcontrol trial. Ann Int Med 119:874-81, 1993).

Formulations

The compounds described above are preferably administered in aformulation including an O-acylated heparin and/or an O-acylated heparintogether with an acceptable carrier for the mode of administration. Anyformulation or drug delivery system containing the active ingredients,which is suitable for the intended use, as are generally known to thoseof skill in the art, can be used. Suitable pharmaceutically acceptablecarriers for oral, rectal, topical or parenteral (including inhaled,subcutaneous, intraperitoneal, intramuscular and intravenous)administration are known to those of skill in the art. The carrier mustbe pharmaceutically acceptable in the sense of being compatible with theother ingredients of the formulation and not deleterious to therecipient thereof.

Formulations suitable for parenteral administration conveniently includesterile aqueous preparation of the active compound which is preferablyisotonic with the blood of the recipient. Thus, such formulations mayconveniently contain distilled water, 5% dextrose in distilled water orsaline. Useful formulations also include concentrated solutions orsolids containing the compound which upon dilution with an appropriatesolvent give a solution suitable for parental administration above.

For enteral administration, a compound can be incorporated into an inertcarrier in discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the active compound;as a powder or granules; or a suspension or solution in an aqueousliquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or adraught. Suitable carriers may be starches or sugars and includelubricants, flavorings, binders, and other materials of the same nature.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface activeor dispersing agents. Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active compound with anysuitable carrier.

A syrup or suspension may be made by adding the active compound to aconcentrated, aqueous solution of a sugar, e.g., sucrose, to which mayalso be added any accessory ingredients. Such accessory ingredients mayinclude flavoring, an agent to retard crystallization of the sugar or anagent to increase the solubility of any other ingredient, e.g., as apolyhydric alcohol, for example, glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppositorywith a conventional carrier, e.g., cocoa butter or Witepsol S55(trademark of Dynamite Nobel Chemical, Germany), for a suppository base.

Alternatively, the compound may be administered in liposomes ormicrospheres (or microparticles). Methods for preparing liposomes andmicrospheres for administration to a patient are well known to those ofskill in the art. U.S. Pat. No. 4,789,734, the contents of which arehereby incorporated by reference, describes methods for encapsulatingbiological materials in liposomes. Essentially, the material isdissolved in an aqueous solution, the appropriate phospholipids andlipids added, along with surfactants if required, and the materialdialyzed or sonicated, as necessary. A review of known methods isprovided by G. Gregoriadis, Chapter 14, “Liposomes,” Drug Carriers inBiology and Medicine, pp. 287-341 (Academic Press, 1979).

Microspheres formed of polymers or proteins are well known to thoseskilled in the art, and can be tailored for passage through thegastrointestinal tract directly into the blood stream. Alternatively,the compound can be incorporated and the microspheres, or composite ofmicrospheres, implanted for slow release over a period of time rangingfrom days to months. See, for example, U.S. Pat. Nos. 4,906,474,4,925,673 and 3,625,214, and Jein, TIPS19:155-157 (1998), the contentsof which are hereby incorporated by reference.

In one embodiment, the O-acylated heparin can be formulated into aliposome or microparticle which is suitably sized to lodge in capillarybeds following intravenous administration. When the liposome ormicroparticle is lodged in the capillary beds surrounding ischemictissue, the agents can be administered locally to the site at which theycan be most effective. Suitable liposomes for targeting ischemic tissueare generally less than about 200 nanometers and are also typicallyunilamellar vesicles, as disclosed, for example, in U.S. Pat. No.5,593,688 to Baldeschweiler, entitled “Liposomal targeting of ischemictissue,” the contents of which are hereby incorporated by reference.

Preferred microparticles are those prepared from biodegradable polymers,such as polyglycolide, polylactide and copolymers thereof. Those ofskill in the art can readily determine an appropriate carrier systemdepending on various factors, including the desired rate of drug releaseand the desired dosage.

In one embodiment, the formulations are administered via catheterdirectly to the inside of blood vessels. The administration can occur,for example, through holes in the catheter. In those embodiments whereinthe active compounds have a relatively long half life (on the order of 1day to a week or more), the formulations can be included inbiodegradable polymeric hydrogels, such as those disclosed in U.S. Pat.No. 5,410,016 to Hubbell et al. These polymeric hydrogels can bedelivered to the inside of a tissue lumen and the active compoundsreleased over time as the polymer degrades. If desirable, the polymerichydrogels can have microparticles or liposomes which include the activecompound dispersed therein, providing another mechanism for thecontrolled release of the active compounds.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active compound intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier or a finely divided solid carrier and then, if necessary,shaping the product into desired unit dosage form.

The formulations can optionally include additional components, such asvarious biologically active substances such as growth factors (includingTGFβ, basic fibroblast growth factor (bFGF), epithelial growth factor(EGF), transforming growth factors alpha and beta (TGFα and TGFβ), nervegrowth factor (NGF), platelet-derived growth factor (PDGF), and vascularendothelial growth factor/vascular permeability factor (VEGF/VPF),antivirals, antibacterials, antiinflammatories, immunosuppressants,analgesics, vascularizing agents, cell adhesion molecules (CAM's), andanticoagulants other than heparin or heparin-like substances.

In addition to the aforementioned ingredients, the formulations mayfurther include one or more optional accessory ingredient(s) utilized inthe art of pharmaceutical formulations, e.g., diluents, buffers,flavoring agents, binders, surface active agents, thickeners,lubricants, suspending agents, preservatives (including antioxidants)and the like.

Finally, compositions of the compound are presented for administrationto the respiratory tract as a snuff or an aerosol or solution for anebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case theparticles of active compound suitably have diameters of less than 50microns, preferably less than 10 microns, more preferably between 2 and5 microns.

Stents, Grafts and Implants

The present invention further provides an intravascular implant coating.The coating includes a therapeutically effective amount of an O-acylatedheparin. The coating can be used in any type of implant. These includeballoon catheters, stents, stent graphs, drug delivery catheters,atherectomy devices, filters, scaffolding devices, anastomothic clips,anastomotic bridges and suture materials.

The coating can also include a polymer matrix, with the polymer being aresorbable polymer selected from the group consisting of poly-α hydroxylacids, polyglycols, polytyrosine carbonates, starch, gelatins,cellulose, and blends and copolymers thereof. Examples of suitablepoly-α hydroxyl acids include polylactides, polyglycol acids, and blendsand co-polymers thereof.

According to the present invention, a coating for an intravascularimplant is provided. The coating can be applied either alone, or withina polymeric matrix, which can be biostable or bioabsorbable, to thesurface of an intravascular device. The coating can be applied directedto the implant or on top of a polymeric substrate, i.e. a primer. Ifdesired, a top coat can be applied to the therapeutic coating.

It should be noted that the present invention relates to a combinatorialtherapy for delivery of more than one agent through a coating on anyintravascular implant. As used herein, implant means any type of medicalor surgical implement, whether temporary or permanent. Delivery can beeither during or after an interventional procedure. Non-limitingexamples of intravascular implants now follow.

The outside surface of a balloon catheter may be coated with the coatingaccording to the present invention and could be released immediately orin a time dependent fashion. When the balloon expands and the wall ofthe vessel is in contact with the balloon, the release of the o-acylatedheparin can begin.

The surface of a stent may be coated with the combination of agents andthe stent is implanted inside the body. The stent struts could be loadedwith several layers of the agents or with just a single layer. Atransporter or a vehicle to load the agents on to the surface can alsobe applied to the stent. The graft material of the stent graft can alsobe coated (in addition to the stent or as an alternative) so that thematerial is transported intravascularly at the site of the location ofthe injury.

The drug delivery catheters that are used to inject drugs and otheragents intravascularly can also be used to deliver the o-acylatedheparins. Other intravascular devices through which the transport canhappen include atherectomy devices, filters, scaffolding devices,anastomotic clips, anastomotic bridges, suture materials etc.

The present invention envisions applying the coating directly to theintravascular implant. However, the coating can be applied to a primer,i.e. a layer or film of material upon which another coating is applied.Furthermore, the o-acylated heparins can be incorporated in a polymermatrix. Polymeric matrices (resorbable and biostable) can be used fordelivery of the therapeutic agents. In some situations, when the agentsare loaded on to the implant, there is a risk of quick erosion of thetherapeutic agents either during the expansion process or during thephase during which the blood flow is at high shear rates at the time ofimplantation. In order to ensure that the therapeutic window of theagents is prolonged over extended periods of time, polymer matrices canbe used.

These polymers could be any one of the following: semitelechelicpolymers for drug delivery, thermo responsive polymeric micelles fortargeted drug delivery, pH or temperature sensitive polymers for drugdelivery, peptide and protein based drug delivery, water insoluble drugcomplex drug delivery matrices polychelating amphiphilic polymers fordrug delivery, bioconjugation of biodegradable poly lactic/glycolic acidfor delivery, elastin mimetic protein networks for delivery, genericallyengineered protein domains for drug delivery, superporbus hydrogelcomposites for drug delivery, interpenetrating polymeric networks fordrug delivery, hyaluronic acid based delivery of drugs, photocrosslinkedpolyanhydrides with controlled hydrolytic delivery, cytokine-includingmacromolecular glycolipids based delivery, cationic polysaccharides fortopical delivery, n-halamine polymer coatings for drug delivery, dextranbased coatings for drug delivery, fluorescent molecules for drugdelivery, self-etching polymerization initiating primes for drugdelivery, and bioactive composites based drug delivery.

One embodiment of the present invention discloses an implant, e.g., astent for implantation into a body, e.g., blood vessel. The implantcomprises a coating of O-acylated heparin or o-acylated heparin incombination with one or more compounds selected from the groupconsisting of (but not limited to) a polymer, fiber polymer,polyurethane, silicone rubber elastomer, drug, hydrogel, or otheracceptable compound or carrier known to those of skill in the art.Methods of coating an implant such as a stent with heparin or heparin incombination with one or more of the compounds listed above, are known tothose of skill in the art and are further described below and in theexamples. Alternatively, O-acylated heparins of the present inventionmay be coated alone or in combination with the above polymer, fiberpolymer, polyurethane, silicone rubber elastomer, drug, hydrogel, orother acceptable compound or carrier known to those of skill in the artonto a bypass graft. The implant, e.g., graft or stent may be used inthe treatment of peripheral artery atherosclerosis disease (PAD).

Whereas the polymer of the coating may be any compatible biostablematerial capable of being adhered to the stent material as a thin layer,hydrophobic materials are preferred because it has been found that therelease of the biologically active species can generally be morepredictably controlled with such materials. Preferred materials includesilicone rubber elastomers and biostable polyurethanes.

Heparin-loaded polymer can be applied by spraying or by dipping thestent graft into a solution or melt, as disclosed, for example, in U.S.Pat. Nos. 5,383,922, 5,824,048, 5,624,411 and 5,733,327. Additionalmethods for providing a drug-loaded polymer are disclosed in U.S. Pat.Nos. 5,637,113 and 5,766,710, where a pre-fabricated film is attached tothe stent. Other methods, such as deposition via photo polymerization,plasma polymerization and the like, are also known in the art and aredescribed in, e.g., U.S. Pat. Nos. 3,525,745, 5,609,629 and 5,824,049and in the below examples.

U.S. Pat. No. 5,549,663 discloses a stent graft having a coating made ofpolyurethane fibers which are applied using conventional wet spinningtechniques. Prior to the covering process, a medication is introducedinto the polymer. Alternatively, a metallic stent cam be coated with apolymeric material and load the polymeric material with a drug.

The Figures have not been drawn to scale, and the dimensions such asdepth and thickness of the various regions and layers have been over orunder emphasized for illustrative purposes. Referring to FIGS. 23A and23B, a stent 10 is formed from a plurality of struts 12. Struts 12 areseparated by gaps 14 and may be interconnected by connecting elements16. Struts 12 can be connected in any suitable configuration and patternto form an a tubular body. While a strut configuration is illustrated,any known stent configuration may be used. Stent 10 is illustratedhaving an outer surface or sidewall 18 (tissue-contacting surface) andan inner surface 20 (blood-contacting surface). A hollow, central bore22 extends longitudinally from a first open end 24 to a second end 26 ofstent 10.

FIG. 24 illustrates stent 10 coated in accordance with the presentinvention. The stent may have a first coating 28 containing anO-acylated heparin on inner surface 20 and/or a second coating 32containing an O-acylated heparin formed on outer surface 18 of stent 10.The coatings can be of any suitable thickness. The thickness of secondcoating 32 can be from about 0.1-15 microns, more narrowly from about 3microns to about 8 microns. By way of example, second coating 32 canhave a thickness of about 4 microns.

Cancer

In another embodiment of the present invention, methods are disclosedfor the treatment and or prevention of cancer. Therapeutic amounts ofO-acylated heparin, particularly O-hexanoylated heparin derivatives andO-butanoylated heparin derivatives are given to a patient alone or incombination with other cancer therapies, known to those of skill in theart. Compounds may be administered before, at the same time as, or afterthe administration of other conventional cancer therapies. O-acylatedheparins of the present invention may be given prior to the diagnosis ofcancer, such as in the case of a patient having a high-risk ofdeveloping cancer, or after the successful treatment of cancer (ie.remission). The compounds of the present invention may also beadministered with the goal of reducing metastases.

Examples of tumors which may be inhibited, but are not limited to, lungcancer (e.g. adenocarcinoma, small cell, and including non-small celllung cancer), pancreatic cancers (e.g. pancreatic carcinoma such as, forexample exocrine pancreatic carcinoma), colon cancers (e.g. colorectalcarcinomas, such as, for example, colon adenocarcinoma and colonadenoma), prostate cancer including the advanced disease, hematopoietictumors of lymphoid lineage (e.g. acute lymphocytic leukemia, B-celllymphoma, Burkitt's lymphoma), myeloid leukemias (for example, acutemyelogenous leukemia (AML)), thyroid follicular cancer, myelodysplasticsyndrome (MDS), tumors of mesenchymal origin (e.g. fibrosarcomas andrhabdomyosarcomas), melanomas, teratocarcinomas, neuroblastomas,gliomas, benign tumor of the skin (e.g. keratoacanthomas), breastcarcinoma (e.g. advanced breast cancer), kidney carcinoma, ovarycarcinoma, bladder carcinoma and epidermal carcinoma.

For the treatment of the above conditions, the compound of the inventionmay be advantageously employed in combination with one or more othermedicinal agents such as anti-cancer agents.

For example, O-acylated heparins of the invention may be given incombination with one or more compounds selected from platinumcoordination compounds for example cisplatin or carboplatin, taxanecompounds for example paclitaxel or docetaxel, camptothecin compoundsfor example irinotecan or topotecan, anti-tumor vinca alkaloids forexample vinblastine, vincristine or vinorelbine, anti-tumor nucleosidederivatives for example 5-fluorouracil, gemcitabine or capecitabine,nitrogen mustard or nitrosourea alkylating agents for examplecyclophosphamide, chlorambucil, carmustine or lomustine, anti-tumoranthracycline derivatives for example daunorubicin, doxorubicin oridarubicin; HER2 antibodies for example trastzumab; and antitumorpodophyllotoxin derivatives for example etoposide or teniposide; andantiestrogen agents including estrogen receptor antagonists or selectiveestrogen receptor modulators preferably tamoxifen, or alternativelytoremifene, droloxifene, faslodex and raloxifene, or aromataseinhibitors such as exemestane, anastrozole, letrazole and vorozole.

Aberrant Smooth Muscle Cell Proliferation

The methods of the present invention can be used to treat disorderswherein smooth muscle cells abnormally proliferate. Such conditionsinclude, but are not limited to, restenosis (following angioplasy,vascular stent placement, coronary artery stent placement, peripheralartery stent placement, or cerebral artery stent placement), pulmonaryhypertension, and pulmonary fibrosis. We have shown that heparin caninhibit fibroblast proliferation (Dahlberg et al. Am Rev. Respir. Dis.143:A357, 1993) and can inhibit pulmonary fibrosis in the rat inresponse to bleomycin. We also have unpublished data showinghexanoylated and butanoylated heparins, which have virtually noanticoagulant property, can also inhibit fibroblast proliferation andthus may offer a potent therapeutic agent for human pulmonary fibrosis.

The methods of the invention provide for the treatment (reduction orcessation) or prevention of disorders wherein smooth muscle cells areabnormally proliferating. These methods include the administration ofO-acylated heparin compounds, preferably O-hexanoylated orO-butanoylated heparin derivatives.

Administration of the compounds of the invention to treat and/or preventaberrant smooth muscle cell proliferation are known to those skilled inthe art and are presented above. Preferably, O-acylated heparin iscoated on an implantable stent, wherein the delivery of the heparin iscontrolled and sufficient to reduce or ablate aberrant smooth musclecell proliferation.

Pulmonary Hypertension and Pulmonary Fibrosis

In yet another embodiment, the present invention is directed to thetreatment and/or prevention of pulmonary hypertension and pulmonaryfibrosis. Preferably, O-acylated heparins of the invention are presentedfor administration to the respiratory tract as a snuff or an aerosol orsolution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase the particles of the active compound suitably have diameters ofless than 50 microns, preferably less than 10 microns, more preferablybetween 2 and 5 microns. The methods of the present invention aredirected to the treatment of both primary and secondary pulmonaryhypertension and pulmonary fibrosis.

Examples Preparation of O-Acylated Heparins

In this example, we describe the synthesis of Low Molecular Weight (LMW)heparin by periodate oxidation, its characterization, and itsO-acylation.

Heparin was fragmented by periodate oxidation based on a modification ofan earlier procedure (described in U.S. Pat. No. 4,990,502), whereinheparin sodium salt (20 g, 1.43 mmol) was dissolved in 175 mL distilledwater. The pH was adjusted to 5.0 using 1 N HCl. NaIO₄ (15 g, 0.070mol), dissolved in 500 mL water, was added in a single portion withstirring. The pH was readjusted to 5.0 using 1 N HCl and left for 24hours at 4° C. in the dark. The solution was dialyzed against 4 volumesof water (with one change of water) for 15 h at 4° C. To theapproximately 1.5 L solution obtained after dialysis, 32 mL of 10 N NaOHwas added. The solution was stirred at room temperature for 3 h. Toprevent the development of colored products, this step was done in thedark.

NaBH₄ (1 g, 0.026 mol) was added in one portion and the approximately1.5 L of solution was stirred for 4 hours. The pH was then adjusted to4.0 using 37% HCl and the solution was stirred for an additional 15 min.The solution was neutralized to pH 7.0 using 1 N NaOH, NaCl (32.8 g,0.56 mol) followed by 2.54 L of ethanol. The solution was left for 3 hwithout stirring and the precipitate was recovered by centrifugation at15000 rpm (22,000×g) for 20 min. The precipitate was recovered bydecantation and suspended in 400 mL absolute ethanol. The solution wasfiltered using a Buchner funnel and left to dry for 5 hours under vacuumaffording 14.2 g of product.

The product was dissolved in 190 mL of water. NaCl (2.8 g, 0.05 mol) wasadded and the pH was adjusted to 3.5 using 1 N HCl. The volume wasadjusted to 280 mL using water. Absolute ethanol (240 mL) was added withstirring. The solution was stirred 15 min and then left without stirringfor 10 hours at room temperature. After decanting, the precipitate wasrecovered and dissolved in water. The ethanol was removed by rotaryevaporation under reduced pressure and freeze-dried affording ˜10 g ofLMW heparin fragments (FIG. 1).

NMR Sample Preparation

For ¹H NMR spectroscopy, approximately 10 mg of each sample wasexchanged by lyophilization three times from 0.5 ml portions of 99.9%²H₂O before being redissolved in ²H₂O for NMR analysis. Chemical shiftsare reported relative to TMS at 0.00 ppm. The degree of substitution(O-acylation) was determined from the ratio of the integrated area ofthe peaks assigned to the aliphatic methyl protons of the hexanoyl group(0.753 ppm) to the anomeric proton of IdoA2S (5.092 ppm) (Table 1, FIG.2).

Gradient PAGE Analysis

Gradient polyacrylamide gel electrophoresis (PAGE) was performed on a 32cm vertical slab gel unit PROTEAN II equipped with Model 1000 powersource from Bio-Rad IRichmond, Calif.). Polyacrylamide linear gradientresolving gels (14×28 cm), 12-22%) total acrylamide) were prepared andrun as previously described (Edens et al., 1992, J. Pharm. Sci. 81,823-827). The molecular sizes of the oligosaccharide samples weredetermined by comparing with a banding ladder of heparin oligosaccharidestandards prepared from bovine lung heparin. Oligosaccharides werevisualized by Alcian blue staining. The average MW of the product wasdetermined to be 6,000.

Anti-Factor Xa and Anti-Factor IIa Activities

LMW heparin and heparin standard were in diluted normal human plasma.Chromogenic Xa substract S-2732(Suc-IIc-Glu(gamma-piperidyl)-Gly-Arg-pNA) 2.9 MM in 50 mM Tris, 7.5 μMEDTA, pH 8.4 buffer (200 μL), was added to 25 μL of plasma containingsample and 200 μL of bovine Factor Xa (1.25/mL). After mixing, thereaction was incubated for 8 min. at 37 degrees Celsius and 200 μL of20% aqueous acetic was added. Residual Factor Xa was then determined bymeasuring absorbance at 405 nm. Anti-factor IIa activity was determinedby incubating 50 mL of LMW heparin in NHP diluted 4-fold with water with50 mL of human thrombin (12 NIH units/mL) at 37° C. for 30 s. then 50 mLof (2.5 mmol/mL of Chromogenic TH (ethylmalonyl-Pro-Aeg-p-nitroanilidehydrochloride) was added, and the amidolytic thrombin activity wasmeasured at 405 nm. Measurements were performed on an ACL 300 plus fromInstrumentation (Lexington, Mass.) and calculated in comparison with USPHeparin Reference Standard (K-3) supplied by U.S. PharmacopeialConvention (Rockville, Md.). The product exhibited no measurableanti-factor Xa or anti-factor IIa activity.

O-Acylated LMW Heparin Derivatives

(1) O-Hexanoyl derivative of periodate-oxidized heparin fragments. Thesewere obtained by treating the tributylaminmonium salt of periodateoxidized heparin fragments with hexanoic anhydride as describedpreviously (Gohda et al., 2001, Biomacromolecules, 2(4):1178-83)(Lormeau U.S. Pat. No. 4,990,502). Briefly, the tributylammonium salt(11.9 g) was dissolved in dry DMF (114 mL), kept under Ar and cooled to0 degrees Celsius. 4-Dimethylaminopyridine (0.695 g, 5.69 mmol),hexanoic anhydride (26.2 mL, 0.113 mol), and tributylamine (227 mL,0.113 mol) were successively added in single portions, and the reactionwas allowed to proceed under argon at room temperature for 24 hours.After cooling to 0° C., 5% NaHCO3 in water (227 mL) was gradually addedand the solution was stirred at room temperature for 48 h. Excess NaHCO3was eliminated by slow, dropwise addition of 1 N HCL (˜200 mL) until pH4was reached and then readjusted to pH 7 with 1 N NaOH (˜150 mL). Colddenatured (95%) ethanol (5 L, 5 vol) was added with stirring. The samplewas allowed to sit overnight at 4 degrees Celsius to afford precipitate.The precipitate was recovered by decanting and dissolved in 0.2 M NaCl(114 mL), and the precipitation procedure was repeated by addingabsolute ethanol (570 mL). The precipitate was recovered bycentrifugation at 15000 rpm for 20 minutes, dissolved in water (114 mL),and passed through a column (300 mL) of Dowez 50WX8(H⁺) cation-exchangeresine and 600 ml was recovered. The acid was neutralized to pH 7 with10 N NaOH and the solution was filtered through a 0.22 μm Milliporefilter. After lyophilization, O-hexanoyl heparin oligosaccharides (7.12g) was obtained as an off-white powder (FIG. 3).

O-Butanoylated LMW Heparin

This derivative was prepared from the tributylaminmonium salt of LMWheparin by treatment with butyric anhydride under the same condition asdescribed for hexanoyl derivative (see above).

Application of Heparin to Stent

O-acylated heparin derivatives can be coated on stents using the methodsset forth in U.S. Pat. No. 6,620,194. The method is generally asfollows.

The application of the coating material to the stent is quite similarfor all of the materials and the same for the heparin and one or moreadditional suspensions prepared as in the above Examples. The suspensionto be applied is transferred to an application device, typically a paintjar attached to an air brush, such as a Badger Model 150, supplied witha source of pressurized air through a regulator (Norgren, 0-160 psi).Once the brush hose is attached to the source of compressed airdownstream of the regulator, the air is applied. The pressure isadjusted to approximately 15-25 psi and the nozzle condition checked bydepressing the trigger.

Any appropriate method can be used to secure the stent for spraying androtating fixtures. Both ends of the relaxed stent can be fastened to thefixture by two resilient retainers, commonly alligator clips, with thedistance between the clips adjusted so that the stent remains in arelaxed, unstretched condition. The rotor is then energized and the spinspeed adjusted to the desired coating speed, nominally about 40 rpm.

With the stent rotating in a substantially horizontal plane, the spraynozzle is adjusted so that the distance from the nozzle to the stent isabout 2-4 inches and the composition is sprayed substantiallyhorizontally with the brush being directed along the stent from thedistal end of the stent to the proximal end and then from the proximalend to the distal end in a sweeping motion at a speed such that onespray cycle occurs in about three stent rotations. Typically a pause ofless than one minute, normally about one-half minute, elapses betweenlayers. Of course, the number of coating layers will vary with theparticular application. For example, for a coating level of 3-4 mg ofheparin per cm.sup.2 of projected area, 20 cycles of coating applicationshould be required and about 30 ml of solution will be consumed for a3.5 mm diameter by 14.5 cm long stent.

The rotation speed of the motor, of course, can be adjusted as can theviscosity of the composition and the flow rate of the spray nozzle asdesired to modify the layered structure. Generally, with the abovemixes, the best results will be obtained at rotational speeds in therange of 30-50 rpm and with a spray nozzle flow rate in the range of4-10 ml of coating composition per minute, depending on the stent size.It is contemplated that a more sophisticated, computer-controlledcoating apparatus will successfully automate the process demonstrated asfeasible in the laboratory.

The coated stent can be thereafter subjected to a curing step in whichthe pre-polymer and crosslinking agents cooperate to produce a curedpolymer matrix containing the biologically active species. The curingprocess involves evaporation of the solvent xylene, THF, etc. and thecuring and crosslinking of the polymer. Certain silicone materials canbe cured at relatively low temperatures, (i.e. RT-50° C.) in what isknown as a room temperature vulcanization (RTV) process. More typically,however, the curing process involves higher temperature curing materialsand the coated stents are put into an oven at approximately 90° C. orhigher for approximately 16 hours. The temperature may be raised to ashigh as 150° C. for dexamethasone containing coated stents. Of course,the time and temperature may vary with particular silicones,crosslinkers, and biologically active species.

Stents coated and cured in the manner described need to be sterilizedprior to packaging for future implantation. For sterilization, gammaradiation is a preferred method particularly for heparin containingcoatings; however, it is possible that stents coated and cured accordingto the process of the invention subjected to gamma sterilization may betoo slow to recover their original posture when delivered to a vascularor other lumen site using a catheter unless a pretreatment step as at 24is first applied to the coated, cured stent.

The pretreatment step can involve an argon plasma treatment of thecoated, cured stent in the unconstrained configuration. In accordancewith this procedure, the stents are placed in a chamber of a plasmasurface treatment system such as a Plasma Science 350 (Himont/PlasmaScience, Foster City, Calif.). The system is equipped with a reactorchamber and RF solid-state generator operating at 13.56 mHz and from0-500 watts power output and being equipped with a microprocessorcontrolled system and a complete vacuum pump package. The reactionchamber contains an unimpeded work volume of 16.75 inches (42.55 cm) by13.5 inches (34.3 cm) by 17.5 inches (44.45 cm) in depth.

In the plasma process, unconstrained coated stents are placed in areactor chamber and the system is purged with nitrogen and a vacuumapplied to 20-50 mTorr. Thereafter, inert gas (argon, helium or mixtureof them) is admitted to the reaction chamber for the plasma treatment. Ahighly preferred method of operation consists of using argon gas,operating at a power range from 200 to 400 watts, a flow rate of 150-650standard ml per minute, which is equivalent to 100-450 mTorr, and anexposure time from 30 seconds to about 5 minutes. The stents can beremoved immediately after the plasma treatment or remain in the argonatmosphere for an additional period of time, typically five minutes.

After this, the stents can be exposed to gamma sterilization at 2.5-3.5Mrad. The radiation may be carried out with the stent in either theradially non-constrained status—or in the radially constrained status.

With respect to the anticoagulant material heparin, the percentage inthe tie layer is nominally from about 20-50% and that of the top layerfrom about 0-30% active material. The coating thickness ratio of the toplayer to the tie layer varies from about 1:10 to 1:2 and is preferablyin the range of from about 1:6 to 1:3.

Suppressing the burst effect also enables a reduction in the drugloading or in other words, allows a reduction in the coating thickness,since the physician will give a bolus injection ofantiplatelet/anticoagulation drugs to the patient during the stentingprocess. As a result, the drug imbedded in the stent can be fully usedwithout waste. Tailoring the first day release, but maximizing secondday and third day release at the thinnest possible coating configurationwill reduce the acute or subacute thrombosis.

RESULTS

Effect on smooth muscle cell proliferation in vitro and in vivo.Hexanoylated LMW heparin significantly inhibited pulmonary artery smoothmuscle cell proliferation in vivo (FIG. 4) and the development ofpulmonary hypertension induced by hypoxia in pig lung (FIG. 5)

In comparison to non-acylated heparin fragments, hexanoylated LMWheparin significantly enhanced the antiproliferative effect of bovinepulmonary artery smooth muscle cells in vitro.

Effect of O-acylation of heparin on tumor growth in vivo. As seen inFIGS. 6-10, butanoylated heparin significantly inhibited the growth ofboth A549 non-small cell lung carcinoma and DMS79 small cell lungcarcinoma in SCID mice (FIG. 6-10). In addition, FIGS. 11 and 12demonstrate that butanoylated heparin significantly inhibited the growthof HCT116 colonic carcinoma in SCID mice.

FIGS. 13 and 14 demonstrate that the above butanoylated heparincompounds exhibit very low anticoagulant effects (compared tonon-acylated controls). Butanoylated heparin had no toxic effect onheart, liver, kidney, and lung of the animals tested (FIG. 15-18).Furthermore, the anti-tumor effect of butanoylated heparin is associatedwith the induction of apoptosis (FIG. 19). The mechanism by whichbutanoylated heparin inhibits tumor growth of lung cancer and coloncancer may involve p27- and p21-RB-E2F pathway (FIG. 20-22). Similarantiproliferative effects were seen with O-hexanoylated LMW heparin onanti-tumor cell growth in vitro.

TABLE 1 Assignment of selected signals in the ¹H NMR spectrum of theO-hexanoyl heparin derivative. Chemical (ppm) shift Residue H-1 H-2 H-3H-4 H-5 GlcNS6S 5.302 3.093 3.539 3.629 3.892 IdoA2S 5.092 4.218 4.0083.920 4.709

1. A composition comprising a hexanoyl derivative of low molecularweight heparin with the chemical structure:

Wherein m=8 and R═CH₃(CH₂)₄—.
 2. A composition comprising a butanoylderivative of low molecular weight heparin with the chemical structure:

wherein m=8 and R═CH₃(CH₂)₂—.
 3. A pharmaceutical compositioncomprising: a) a hexanoyl derivative of low molecular weight heparinwith the chemical structure:

wherein m=8 and R═CH₃(CH₂)₄—; and b) a pharmaceutically acceptablecarrier.
 4. A pharmaceutical composition comprising: a) a butanoylderivative of low molecular weight heparin with the chemical structure:

wherein m=8 and R═CH₃(CH₂)₂—; and b) a pharmaceutically acceptablecarrier.
 5. The pharmaceutical composition of claim 3, which isformulated for oral, rectal, topical or parenteral administration.
 6. Acomposition for intravascular implant coating comprising a hexanoylderivative of low molecular weight heparin with the chemical structure:

wherein m=8 and R═CH₃(CH₂)₄—.
 7. A composition for intravascular implantcoating comprising a butanoyl derivative of low molecular weight heparinwith the chemical structure:

wherein m=8 and R═CH₃(CH₂)₂—.
 8. A polymer matrix which has incorporatedtherein, a hexanoyl derivative of low molecular weight heparin with thechemical structure:

wherein m=8 and R═CH₃(CH₂)₄—.
 9. A polymer matrix which has incorporatedtherein, a butanoyl derivative of low molecular weight heparin with thechemical structure:

wherein m=8 and R═CH₃(CH₂)₂—.
 10. The pharmaceutical composition ofclaim 4, which is formulated for oral, rectal, topical or parenteraladministration.